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1.
Genet Epidemiol ; 43(6): 704-716, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31172578

RESUMO

Phenotypic heterogeneity is a hallmark of complex traits, and genetic studies of such traits may focus on them as a single diagnostic entity or by analyzing specific components. For example, in orofacial clefting (OFC), three subtypes-cleft lip (CL), cleft lip and palate (CLP), and cleft palate (CP) have been studied separately and in combination. To further dissect the genetic architecture of OFCs and how a given associated locus may be contributing to distinct subtypes of a trait we developed a framework for quantifying and interpreting evidence of subtype-specific or shared genetic effects in complex traits. We applied this technique to create a "cleft map" of the association of 30 genetic loci with three OFC subtypes. In addition to new associations, we found loci with subtype-specific effects (e.g., GRHL3 [CP], WNT5A [CLP]), as well as loci associated with two or all three subtypes. We cross-referenced these results with mouse craniofacial gene expression datasets, which identified additional promising candidate genes. However, we found no strong correlation between OFC subtypes and expression patterns. In aggregate, the cleft map revealed that neither subtype-specific nor shared genetic effects operate in isolation in OFC architecture. Our approach can be easily applied to any complex trait with distinct phenotypic subgroups.

2.
Hum Genet ; 137(11-12): 941-954, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30417254

RESUMO

Isolated or syndromic congenital cataracts are heterogeneous developmental defects, making the identification of the associated genes challenging. In the past, mouse lens expression microarrays have been successfully applied in bioinformatics tools (e.g., iSyTE) to facilitate human cataract-associated gene discovery. To develop a new resource for geneticists, we report high-throughput RNA sequencing (RNA-seq) profiles of mouse lens at key embryonic stages (E)10.5 (lens pit), E12.5 (primary fiber cell differentiation), E14.5 and E16.5 (secondary fiber cell differentiation). These stages capture important events as the lens develops from an invaginating placode into a transparent tissue. Previously, in silico whole-embryo body (WB)-subtraction-based "lens-enriched" expression has been effective in prioritizing cataract-linked genes. To apply an analogous approach, we generated new mouse WB RNA-seq datasets and show that in silico WB subtraction of lens RNA-seq datasets successfully identifies key genes based on lens-enriched expression. At ≥2 counts-per-million expression, ≥1.5 log2 fold-enrichment (p < 0.05) cutoff, E10.5 lens exhibits 1401 enriched genes (17% lens-expressed genes), E12.5 lens exhibits 1937 enriched genes (22% lens-expressed genes), E14.5 lens exhibits 2514 enriched genes (31% lens-expressed genes), and E16.5 lens exhibits 2745 enriched genes (34% lens-expressed genes). Biological pathway analysis identified genes associated with lens development, transcription regulation and signaling pathways, among other functional groups. Furthermore, these new RNA-seq data confirmed high expression of established cataract-linked genes and identified new potential regulators in the lens. Finally, we developed new lens stage-specific UCSC Genome Brower annotation tracks and made these publicly accessible through iSyTE ( https://research.bioinformatics.udel.edu/iSyTE/ ) for user-friendly visualization of lens gene expression/enrichment to prioritize genes from high-throughput data from cataract cases.

3.
Eur J Med Genet ; 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30472488

RESUMO

The SPECC1L protein plays a role in adherens junctions involved in cell adhesion, actin cytoskeleton organization, microtubule stabilization, spindle organization and cytokinesis. It modulates PI3K-AKT signaling and controls cranial neural crest cell delamination during facial morphogenesis. SPECC1L causative variants were first identified in individuals with oblique facial clefts. Recently, causative variants in SPECC1L were reported in a pedigree reported in 1988 as atypical Opitz GBBB syndrome. Six families with SPECC1L variants have been reported thus far. We report here eight further pedigrees with SPECC1L variants, including a three-generation family, and a further individual of a previously published family. We discuss the nosology of Teebi and GBBB, and the syndromes related to SPECC1L variants. Although the phenotype of individuals with SPECC1L mutations shows overlap with Opitz syndrome in its craniofacial anomalies, the canonical laryngeal malformations and male genital anomalies are not observed. Instead, individuals with SPECCL1 variants have branchial fistulae, omphalocele, diaphragmatic hernias, and uterus didelphis. We also point to the clinical overlap of SPECC1L syndrome with mild Baraitser-Winter craniofrontofacial syndrome: they share similar dysmorphic features (wide, short nose with a large tip, cleft lip and palate, blepharoptosis, retrognathia, and craniosynostosis), although intellectual disability, neuronal migration defect, and muscular problems remain largely specific to Baraitser-Winter syndrome. In conclusion, we suggest that patients with pathogenic variants in SPECC1L should not be described as "dominant (or type 2) Opitz GBBB syndrome", and instead should be referred to as "SPECC1L syndrome" as both disorders show distinctive, non overlapping developmental anomalies beyond facial communalities.

4.
Birth Defects Res ; 109(1): 27-37, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-28029220

RESUMO

BACKGROUND: Recent advances in genomics methodologies, in particular the availability of next-generation sequencing approaches have made it possible to identify risk loci throughout the genome, in particular the exome. In the current study, we present findings from an exome study conducted in five affected individuals of a multiplex family with cleft palate only. METHODS: The GEnome MINIng (GEMINI) pipeline was used to functionally annotate the single nucleotide polymorphisms, insertions and deletions. Filtering methods were applied to identify variants that are clinically relevant and present in affected individuals at minor allele frequencies (≤1%) in the 1000 Genomes Project single nucleotide polymorphism database, Exome Aggregation Consortium, and Exome Variant Server databases. The bioinformatics tool Systems Tool for Craniofacial Expression-Based Gene Discovery was used to prioritize cleft candidates in our list of variants, and Sanger sequencing was used to validate the presence of identified variants in affected and unaffected relatives. RESULTS: Our analyses approach narrowed the candidates down to the novel missense variant in ARHGAP29 (GenBank: NM_004815.3, NP_004806.3;c.1654T>C [p.Ser552Pro]. A functional assay in zebrafish embryos showed that the encoded protein lacks the activity possessed by its wild-type counterpart, and migration assays revealed that keratinocytes transfected with wild-type ARHGAP29 migrated faster than counterparts transfected with the p.Ser552Pro ARHGAP29 variant or empty vector (control). CONCLUSION: These findings reveal ARHGAP29 to be a regulatory protein essential for proper development of the face, identifies an amino acid that is key for this, and provides a potential new diagnostic tool.Birth Defects Research 109:27-37, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Fissura Palatina/genética , Proteínas Ativadoras de GTPase/genética , Alelos , Animais , Fenda Labial/genética , Biologia Computacional , Modelos Animais de Doenças , Exoma , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Frequência do Gene/genética , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Exoma , Peixe-Zebra/embriologia , Peixe-Zebra/genética
5.
Sci Rep ; 6: 17735, 2016 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-26787558

RESUMO

Cranial neural crest cells (CNCCs) delaminate from embryonic neural folds and migrate to pharyngeal arches, which give rise to most mid-facial structures. CNCC dysfunction plays a prominent role in the etiology of orofacial clefts, a frequent birth malformation. Heterozygous mutations in SPECC1L have been identified in patients with atypical and syndromic clefts. Here, we report that in SPECC1L-knockdown cultured cells, staining of canonical adherens junction (AJ) components, ß-catenin and E-cadherin, was increased, and electron micrographs revealed an apico-basal diffusion of AJs. To understand the role of SPECC1L in craniofacial morphogenesis, we generated a mouse model of Specc1l deficiency. Homozygous mutants were embryonic lethal and showed impaired neural tube closure and CNCC delamination. Staining of AJ proteins was increased in the mutant neural folds. This AJ defect is consistent with impaired CNCC delamination, which requires AJ dissolution. Further, PI3K-AKT signaling was reduced and apoptosis was increased in Specc1l mutants. In vitro, moderate inhibition of PI3K-AKT signaling in wildtype cells was sufficient to cause AJ alterations. Importantly, AJ changes induced by SPECC1L-knockdown were rescued by activating the PI3K-AKT pathway. Together, these data indicate SPECC1L as a novel modulator of PI3K-AKT signaling and AJ biology, required for neural tube closure and CNCC delamination.


Assuntos
Junções Aderentes/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Fosfoproteínas/deficiência , Animais , Apoptose/genética , Biomarcadores , Moléculas de Adesão Celular/metabolismo , Linhagem da Célula/genética , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos , Modelos Biológicos , Mutação , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/patologia , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
6.
J Med Genet ; 52(2): 104-10, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25412741

RESUMO

BACKGROUND: Opitz G/BBB syndrome is a heterogeneous disorder characterised by variable expression of midline defects including cleft lip and palate, hypertelorism, laryngealtracheoesophageal anomalies, congenital heart defects, and hypospadias. The X-linked form of the condition has been associated with mutations in the MID1 gene on Xp22. The autosomal dominant form has been linked to chromosome 22q11.2, although the causative gene has yet to be elucidated. METHODS AND RESULTS: In this study, we performed whole exome sequencing on DNA samples from a three-generation family with characteristics of Opitz G/BBB syndrome with negative MID1 sequencing. We identified a heterozygous missense mutation c.1189A>C (p.Thr397Pro) in SPECC1L, located at chromosome 22q11.23. Mutation screening of an additional 19 patients with features of autosomal dominant Opitz G/BBB syndrome identified a c.3247G>A (p.Gly1083Ser) mutation segregating with the phenotype in another three-generation family. CONCLUSIONS: Previously, SPECC1L was shown to be required for proper facial morphogenesis with disruptions identified in two patients with oblique facial clefts. Collectively, these data demonstrate that SPECC1L mutations can cause syndromic forms of facial clefting including some cases of autosomal dominant Opitz G/BBB syndrome and support the original linkage to chromosome 22q11.2.


Assuntos
Proteínas de Ligação ao Cálcio/química , Esôfago/anormalidades , Genes Dominantes , Predisposição Genética para Doença , Hipertelorismo/genética , Hipospadia/genética , Proteínas dos Microfilamentos/química , Mutação/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Adulto , Sequência de Bases , Análise Mutacional de DNA , Éxons/genética , Família , Feminino , Testes Genéticos , Humanos , Lactente , Masculino , Proteínas dos Microtúbulos/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Linhagem , Fenótipo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética
7.
Hematol Oncol Stem Cell Ther ; 8(1): 6-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25482588

RESUMO

Single or multilineage bone marrow failure can be a serious health problem caused by hereditary and non-hereditary causes such as exposure to drugs or environmental toxins. Normal hematopoiesis requires the integrity of several pathways including the THPO-MPL pathway. Over the last two decades, significant advances in the understanding of normal and abnormal functions of this and related pathways have led to novel diagnostic and therapeutic options.


Assuntos
Medula Óssea/fisiologia , Hematopoese/fisiologia , Transdução de Sinais/fisiologia , Trombopoetina/metabolismo , Humanos
8.
Eur J Med Genet ; 57(2-3): 76-80, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24462885

RESUMO

Recently, 3 unrelated children with a potentially novel 3q26.33-3q27.2 microdeletion syndrome were reported. We now report a new 9 ½ years old Caucasian boy with a 2 Mb deletion of the same genomic region in combination with Klinefelter syndrome. He presented with facial dysmorphism, developmental delay, Asperger syndrome, thrombocytopenia, recurrent infections and hypogammaglobulinemia. The deletion in our patient improves upon the minimum region of the novel 3q26.33-3q27.2 microdeletion, and provides additional insights into the underlying genetic basis of the observed phenotypes. Consistent with two of three previously described patients, our patient also presents with thrombocytopenia, which we postulate is caused by haploinsufficiency of THPO. In addition, haploinsufficiency of LAMP3, a lymphoid and dendritic cell expressed protein that is implicated in bacterial and viral infections, pulmonary surfactant protein transport and amelogenin degradation, may be a novel cause for the immune deficiency, lung disease and dental abnormalities respectively as seen in these patients.


Assuntos
Deleção Cromossômica , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 3 , Criança , Hibridização Genômica Comparativa , Facies , Humanos , Hibridização in Situ Fluorescente , Masculino , Síndrome
9.
Blood ; 122(20): 3440-9, 2013 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-24085763

RESUMO

We recently identified 2 siblings afflicted with idiopathic, autosomal recessive aplastic anemia. Whole-exome sequencing identified a novel homozygous missense mutation in thrombopoietin (THPO, c.112C>T) in both affected siblings. This mutation encodes an arginine to cysteine substitution at residue 38 or residue 17 excluding the 21-amino acid signal peptide of THPO receptor binding domain (RBD). THPO has 4 conserved cysteines in its RBD that form 2 disulfide bonds. Our in silico modeling predicts that introduction of a fifth cysteine may disrupt normal disulfide bonding to cause poor receptor binding. In functional assays, the mutant-THPO-containing media shows two- to threefold reduced ability to sustain UT7-TPO cells, which require THPO for proliferation. Both parents and a sibling with heterozygous R17C change have reduced platelet counts, whereas a sibling with wild-type sequence has normal platelet count. Thus, the R17C partial loss-of-function allele results in aplastic anemia in the homozygous state and mild thrombocytopenia in the heterozygous state in our family. Together with the recent identification of THPO receptor (MPL) mutations and the effects of THPO agonists in aplastic anemia, our results have clinical implications in the diagnosis and treatment of patients with aplastic anemia and highlight a role for the THPO-MPL pathway in hematopoiesis in vivo.


Assuntos
Anemia Aplástica/genética , Exoma/genética , Trombopoetina/genética , Adolescente , Adulto , Substituição de Aminoácidos , Anemia Aplástica/tratamento farmacológico , Sequência de Bases , Células Cultivadas , Criança , Clonagem Molecular , Hibridização Genômica Comparativa , Cistina/química , Éxons/genética , Feminino , Genes Recessivos , Genótipo , Humanos , Masculino , Micronésia , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Terapia de Alvo Molecular , Mutação de Sentido Incorreto , Linhagem , Ligação Proteica , Conformação Proteica , Receptores de Trombopoetina/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-Atividade , Trombopoetina/química , Trombopoetina/metabolismo , Adulto Jovem
10.
Development ; 140(13): 2697-702, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23720046

RESUMO

Bmp4 expression is tightly regulated during embryonic tooth development, with early expression in the dental epithelial placode leading to later expression in the dental mesenchyme. Msx1 is among several transcription factors that are induced by epithelial Bmp4 and that, in turn, are necessary for the induction and maintenance of dental mesenchymal Bmp4 expression. Thus, Msx1(-/-) teeth arrest at early bud stage and show loss of Bmp4 expression in the mesenchyme. Ectopic expression of Bmp4 rescues this bud stage arrest. We have identified Tbx2 expression in the dental mesenchyme at bud stage and show that this can be induced by epithelial Bmp4. We also show that endogenous Tbx2 and Msx1 can physically interact in mouse C3H10T1/2 cells. In order to ascertain a functional relationship between Msx1 and Tbx2 in tooth development, we crossed Tbx2 and Msx1 mutant mice. Our data show that the bud stage tooth arrest in Msx1(-/-) mice is partially rescued in Msx1(-/-);Tbx2(+/-) compound mutants. This rescue is accompanied by formation of the enamel knot (EK) and by restoration of mesenchymal Bmp4 expression. Finally, knockdown of Tbx2 in C3H10T1/2 cells results in an increase in Bmp4 expression. Together, these data identify a novel role for Tbx2 in tooth development and suggest that, following their induction by epithelial Bmp4, Msx1 and Tbx2 in turn antagonistically regulate odontogenic activity that leads to EK formation and to mesenchymal Bmp4 expression at the key bud-to-cap stage transition.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Fator de Transcrição MSX1/metabolismo , Proteínas com Domínio T/metabolismo , Dente/embriologia , Dente/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Linhagem Celular , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Fator de Transcrição MSX1/genética , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Mutantes , Odontogênese/genética , Odontogênese/fisiologia , Ligação Proteica , Proteínas com Domínio T/genética
11.
J Biol Chem ; 288(18): 12580-95, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23515314

RESUMO

Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and ß-catenin. Lef-1, ß-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/fisiologia , Animais , Células CHO , Núcleo Celular/genética , Cricetinae , Cricetulus , Proteínas de Homeodomínio/genética , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Complexos Multiproteicos/genética , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/genética , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , beta Catenina/genética , beta Catenina/metabolismo
12.
Hum Genet ; 131(2): 235-50, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21769484

RESUMO

We describe a male patient (patient DGAP113) with a balanced translocation, 46,XY,t(1;3)(q31.3;q13.13), severe bilateral congenital cataracts, CNS abnormalities and mild developmental delay. Fluorescence in situ hybridization (FISH) and suppression PCR demonstrated that the chromosome 3 breakpoint lies ~515 kb upstream of the PVRL3 gene, while the chromosome 1 breakpoint lies ~50 kb upstream of the NEK7 gene. Despite the fact that NEK7 is closer to a translocation breakpoint than PVRL3, NEK7 transcript levels are unaltered in patient DGAP113 lymphoblastoid cells and Nek7-deficient mice exhibit no detectable ocular phenotype. In contrast, the expression of PVRL3, which encodes the cell adhesion protein Nectin 3, is significantly reduced in patient DGAP113 lymphoblastoid cells, likely due to a position effect caused by the chromosomal translocation. Nectin 3 is expressed in the mouse embryonic ciliary body and lens. Moreover, Pvrl3 knockout mice as well as a spontaneous mouse mutant ari (anterior retinal inversion), that maps to the Pvrl3 locus, exhibit lens and other ocular defects involving the ciliary body. Collectively, these data identify PVRL3 as a critical gene involved in a Nectin-mediated cell-cell adhesion mechanism in human ocular development.


Assuntos
Catarata/congênito , Catarata/genética , Moléculas de Adesão Celular/genética , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Quebra Cromossômica , Humanos , Linfócitos , Masculino , Camundongos , Mutação , Quinases Relacionadas a NIMA , Nectinas , Proteínas Serina-Treonina Quinases/metabolismo , Translocação Genética
13.
Am J Hum Genet ; 89(1): 44-55, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21703590

RESUMO

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Specc1l is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.


Assuntos
Fissura Palatina/genética , Disostose Craniofacial/genética , Proteínas do Citoesqueleto/deficiência , Anormalidades do Olho/genética , Anormalidades Maxilofaciais/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Actinas/genética , Animais , Adesão Celular , Linhagem Celular , Movimento Celular/genética , Proliferação de Células , Fissura Palatina/patologia , Disostose Craniofacial/patologia , Drosophila/genética , Drosophila/metabolismo , Anormalidades do Olho/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Hibridização In Situ , Masculino , Anormalidades Maxilofaciais/patologia , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Tubulina (Proteína)/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
14.
Science ; 331(6024): 1571-6, 2011 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-21436445

RESUMO

The precise transcriptional regulation of gene expression is essential for vertebrate development, but the role of posttranscriptional regulatory mechanisms is less clear. Cytoplasmic RNA granules (RGs) function in the posttranscriptional control of gene expression, but the extent of RG involvement in organogenesis is unknown. We describe two human cases of pediatric cataract with loss-of-function mutations in TDRD7 and demonstrate that Tdrd7 nullizygosity in mouse causes cataracts, as well as glaucoma and an arrest in spermatogenesis. TDRD7 is a Tudor domain RNA binding protein that is expressed in lens fiber cells in distinct TDRD7-RGs that interact with STAU1-ribonucleoproteins (RNPs). TDRD7 coimmunoprecipitates with specific lens messenger RNAs (mRNAs) and is required for the posttranscriptional control of mRNAs that are critical to normal lens development and to RG function. These findings demonstrate a role for RGs in vertebrate organogenesis.


Assuntos
Catarata/genética , Regulação da Expressão Gênica no Desenvolvimento , Glaucoma/genética , Cristalino/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Catarata/congênito , Catarata/patologia , Linhagem Celular , Embrião de Galinha , Cristalinas/genética , Cristalinas/metabolismo , Grânulos Citoplasmáticos/metabolismo , Desenvolvimento Embrionário , Feminino , Técnicas de Silenciamento de Genes , Humanos , Hipospadia/genética , Cristalino/embriologia , Masculino , Camundongos , Mutação , Organogênese , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Espermatogênese/genética
15.
Development ; 136(11): 1939-49, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19429790

RESUMO

The ablation of Apc function or the constitutive activation of beta-catenin in embryonic mouse oral epithelium results in supernumerary tooth formation, but the underlying mechanisms and whether adult tissues retain this potential are unknown. Here we show that supernumerary teeth can form from multiple regions of the jaw and that they are properly mineralized, vascularized, innervated and can start to form roots. Even adult dental tissues can form new teeth in response to either epithelial Apc loss-of-function or beta-catenin activation, and the effect of Apc deficiency is mediated by beta-catenin. The formation of supernumerary teeth via Apc loss-of-function is non-cell-autonomous. A small number of Apc-deficient cells is sufficient to induce surrounding wild-type epithelial and mesenchymal cells to participate in the formation of new teeth. Strikingly, Msx1, which is necessary for endogenous tooth development, is dispensable for supernumerary tooth formation. In addition, we identify Fgf8, a known tooth initiation marker, as a direct target of Wnt/beta-catenin signaling. These studies identify key mechanistic features responsible for supernumerary tooth formation.


Assuntos
Proteína da Polipose Adenomatosa do Colo/fisiologia , Dente Supranumerário/embriologia , Proteínas Wnt/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Células Cultivadas , Desenvolvimento Embrionário , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Dente Supranumerário/metabolismo , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo
16.
Am J Hum Genet ; 82(3): 712-22, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18319076

RESUMO

Apparently balanced chromosomal rearrangements in individuals with major congenital anomalies represent natural experiments of gene disruption and dysregulation. These individuals can be studied to identify novel genes critical in human development and to annotate further the function of known genes. Identification and characterization of these genes is the goal of the Developmental Genome Anatomy Project (DGAP). DGAP is a multidisciplinary effort that leverages the recent advances resulting from the Human Genome Project to increase our understanding of birth defects and the process of human development. Clinically significant phenotypes of individuals enrolled in DGAP are varied and, in most cases, involve multiple organ systems. Study of these individuals' chromosomal rearrangements has resulted in the mapping of 77 breakpoints from 40 chromosomal rearrangements by FISH with BACs and fosmids, array CGH, Southern-blot hybridization, MLPA, RT-PCR, and suppression PCR. Eighteen chromosomal breakpoints have been cloned and sequenced. Unsuspected genomic imbalances and cryptic rearrangements were detected, but less frequently than has been reported previously. Chromosomal rearrangements, both balanced and unbalanced, in individuals with multiple congenital anomalies continue to be a valuable resource for gene discovery and annotation.


Assuntos
Quebra Cromossômica , Anormalidades Congênitas/genética , Genoma Humano/genética , Desenvolvimento Humano , Mapeamento Cromossômico , Projeto Genoma Humano , Humanos
17.
Science ; 313(5794): 1751, 2006 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-16990542

RESUMO

The posttranslational modification sumoylation can have multiple effects on its substrate proteins. We studied a patient with isolated cleft lip and palate and a balanced chromosomal translocation that disrupts the SUMO1 (small ubiquitin-related modifier) gene, resulting in haploinsufficiency. In mouse, we found that Sumo1 is expressed in the developing lip and palate and that a Sumo1 hypomorphic allele manifests an incompletely penetrant orofacial clefting phenotype. Products of several genes implicated in clefting are sumoylated, and the Sumo1 hypomorphic allele interacts genetically with a loss-of-function allele for one of these loci. Thus, sumoylation defines a network of genes important for palatogenesis.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Adulto , Animais , Linhagem Celular , Pré-Escolar , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cariotipagem , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Palato/embriologia , Palato/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteína SUMO-1/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Células-Tronco/metabolismo , Translocação Genética
18.
Birth Defects Res A Clin Mol Teratol ; 76(3): 175-81, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16498627

RESUMO

BACKGROUND: Mutations in the PITX2 homeobox gene are known to contribute to Axenfeld-Rieger syndrome (ARS), an autosomal-dominant developmental disorder. Although most mutations are in the homeodomain and result in a loss of function, there is a growing subset in the C-terminal domain that has not yet been characterized. These mutations are of particular interest because the C-terminus has both inhibitory and stimulatory activities. METHODS: In this study we used a combination of in vitro DNA binding and transfection reporter assays to investigate the fundamental issue of whether C-terminal mutations result in gain or loss of function at a cellular level. RESULTS: We report a new frameshift mutation in the PITX2 allele that predicts a truncated protein lacking most of the C-terminal domain (D122FS). This newly reported mutant and another ARS C-terminal mutant (W133Stop) both have greater binding than wild-type to the bicoid element. Of interest, the mutants yielded approximately 5-fold greater activation of the prolactin promoter in CHO cells, even though the truncated proteins were expressed at lower levels than the wild-type protein. The truncated proteins also had greater than wild-type activity in 2 other cell lines, including the LS8 oral epithelial line that expresses the endogenous Pitx2 gene. CONCLUSIONS: The results indicate that the PITX2 C-terminal domain has inhibitory activity and support the notion that ARS may also be caused by gain-of-function mutations.


Assuntos
Anormalidades Múltiplas/genética , Segmento Anterior do Olho/anormalidades , Proteínas de Homeodomínio/genética , Mutação , Anormalidades Dentárias/genética , Fatores de Transcrição/genética , Cordão Umbilical/anormalidades , Anormalidades Múltiplas/metabolismo , Animais , Células CHO , Pré-Escolar , Cricetinae , Cricetulus , Análise Mutacional de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Feminino , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Síndrome , Anormalidades Dentárias/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia
19.
J Biol Chem ; 279(50): 52087-94, 2004 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-15466416

RESUMO

The PITX2 homeodomain protein is mutated in patients with Axenfeld-Rieger syndrome and is involved in the development of multiple organ systems, including the heart. We have examined the interaction of PITX2 isoforms with myocyte-enhancing factor 2A (MEF2A), which is a known regulator of cardiac development. A direct interaction between PITX2a and MEF2A was demonstrated using yeast two-hybrid and GST pull-down assays. To study the functional significance of this interaction, we used the atrial natriuretic factor (ANF) promoter. Coexpression of MEF2A and PITX2a or Pitx2c resulted in a strong synergistic activation of the ANF promoter in LS8 oral epithelial cells but not in other cell lines (NIH/3T3, Chinese hamster ovary, or C2C12). The synergism was dependent on promoter context, because it required MEF2 binding sites and was not seen with two other PITX2 target promoters. DNA binding by MEF2A was required but not sufficient for synergism. Upstream activators of p38 MAP kinases, MKK3 and MKK6, increased PITX2a and Pitx2c activity to yield up to 90-fold activation of the ANF promoter in LS8 cells. Because Axenfeld-Rieger syndrome is autosomal dominant and affects development of the oral epithelium, we tested one of the known PITX2 mutants. The PITX2a-K88E mutant protein suppressed wild type PITX2a synergism with MEF2A. These results demonstrate a promoter- and cell-specific functional interaction between PITX2 and MEF2A and suggest the possibility of coordinate control by these factors in the oral epithelium.


Assuntos
Fator Natriurético Atrial/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Células CHO , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Técnicas In Vitro , Proteínas de Domínio MADS , Fatores de Transcrição MEF2 , Camundongos , Mioblastos/metabolismo , Fatores de Regulação Miogênica , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Mol Cell Biol ; 23(6): 1968-82, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12612071

RESUMO

Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.


Assuntos
Genes Dominantes , Genes Homeobox , Proteínas de Homeodomínio/química , Proteínas Nucleares , Fatores de Transcrição/química , Anormalidades Múltiplas/genética , Substituição de Aminoácidos , Animais , Células CHO , Células Cultivadas , Códon/genética , Anormalidades Craniofaciais/genética , Cricetinae , Cricetulus , DNA/metabolismo , DNA Complementar/genética , Dimerização , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Glaucoma/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Oligodesoxirribonucleotídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Síndrome , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido
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